Michel A. Hoogenkamp

Effect of Psidium cattleianum leaf extract on Streptococcus mutans viability, protein expression and acid production.

Plants naturally produce secondary metabolites that can be used as antimicrobials. The aim of this study was to assess the effects of Psidium cattleianum leaf extract on Streptococcus mutans. The extract (100%) was obtained by decoction of 100 g of leaves in 600 ml of deionized water. To assess killing, S. mutans biofilms were treated with water (negative control) or various extract dilutions [100, 50, 25% (v/v) in water] for 5 or 60 min. To evaluate the effect on protein expression, biofilms were exposed to water or 1.6% (v/v) extract for 120 min, proteins were extracted and submitted to 2-dimensional difference gel electrophoresis. Differentially expressed proteins were identified by mass spectrometry. The effect of 1.6% (v/v) extract on acid production was determined by pH measurements and compared to a water control. Viability was similar after 5 min of treatment with the 100% extract or 60 min with the 50% extract (about 0.03% survival). There were no differences in viability between the biofilms exposed to the 25 or 50% extract after 60 min of treatment (about 0.02% survival). Treatment with the 1.6% extract significantly changed protein expression. The abundance of 24 spots was decreased compared to water (p < 0.05). The extract significantly inhibited acid production (p < 0.05). It is concluded that P. cattleianum leaf extract kills S. mutans grown in biofilms when applied at high concentrations. At low concentrations it inhibits S. mutans acid production and reduces the expression of proteins involved in general metabolism, glycolysis and lactic acid production.

Influence of Streptococcus mutans on Enterococcus faecalis Biofilm Formation

INTRODUCTION An important virulence factor of Enterococcus faecalis is its ability to form biofilms. Most studies on biofilm formation have been carried out by using E. faecalis monocultures. Given the polymicrobial nature of root canal infections, it is important to understand biofilm formation of E. faecalis in the presence of other microorganisms. METHODS Eight clinical strains of E. faecalis were tested for biofilm formation on hydroxyapatite disks in the presence and absence of a Streptococcus mutans biofilm. RESULTS Significantly more E. faecalis viable cells were found in biofilms in the presence of S. mutans. This phenomenon was, however, strain-dependent. Of the 8 strains tested, biofilm formation of strains AA-OR34, ER5/1, and V583 was not influenced by S. mutans biofilms. CONCLUSIONS The results from this study, especially the strain difference, underline the importance of studying biofilm formation in a more realistic multispecies setting.

The effect of chemotherapeutic agents on titanium-adherent biofilms

OBJECTIVE To assess the effectiveness of different chemotherapeutic agents on biofilm-contaminated titanium surfaces. MATERIAL AND METHODS This study used a recently described biofilm model. In experiment 1, Streptococcus mutans biofilms grown on titanium discs were treated with (1) EDTA, (2) citric acid (CA), (3) cetylpyridium chloride, (4) Ardox-X, (5) hydrogen peroxide (H(2) O(2) ), (6) chlorhexidine (CHX) and (7) water. In experiment 2, polymicrobial biofilms were treated with (1) CA, (2) Ardox-X, (3) H(2) O(2) , (4) Ardox-X followed by CA, (5) H(2) O(2) followed by CA, (6) CHX and (7) water. Aliquots of the suspended biofilms were plated and incubated anaerobically to enable counts of the total remaining viable bacteria, which were expressed as CFUs. Following incubation, the amount of protein remaining in the treated S. mutans biofilms was quantified to assess the removal potency of each treatment agent. RESULTS H(2) O(2) , Ardox-X and CA killed significantly more S. mutans compared with the other treatments. H(2) O(2) and CA removed significantly more protein than water. CA and the combination treatments were significantly more effective against the polymicrobial biofilms than CHX, H(2) O(2) and Ardox-X. The difference in the killing efficacy between CA alone and the combination treatments was not statistically significant. CONCLUSION Among the chemicals tested, CA demonstrated the greatest decontamination capacity with respect to both the killing and the removal of biofilm cells. This combination of effects is clinically desirable because it promotes biocompatibility and healing around a previously contaminated implant surface. These results should, however, be validated in in vivo studies.

Resazurin Metabolism Assay for Root Canal Disinfectant Evaluation on Dual-species Biofilms

INTRODUCTION Endodontic infections are caused by polymicrobial biofilms. Therefore, novel root canal disinfectants should be evaluated not only on single-species biofilms but also on dual- or mixed-species biofilms. A simple, high-throughput assay is urgently needed for this. In this study, the application of the resazurin metabolism assay was investigated for the evaluation of a root canal disinfectant on dual-species biofilms. METHODS Enterococcus faecalis with or without Streptococcus mutans in biofilms were formed in an active attachment biofilm model for 24 hours. Subsequently, the biofilms were treated with various concentrations of NaOCl for 1 minute. After resazurin metabolism by both organisms was confirmed, treatment efficacies using 0.0016% resazurin were evaluated. RESULTS During NaOCl treatments, resazurin metabolism displays a clear dose response, not only in single-species E. faecalis (or S. mutans) biofilms but also in dual-species biofilms. Notably, the assay revealed that the resistance of dual-species biofilms to NaOCl was 30-fold higher than in single-species E. faecalis biofilms. Viability counts on a selected NaOCl treatment (0.004%) confirmed this result and showed the increased resistance of E. faecalis in dual-species biofilms. CONCLUSIONS Clearly, the high-throughput and low cost resazurin metabolism assay has a great potential for testing novel root canal antimicrobial agents in mixed-species biofilms.

The Effects of Fractions from Shiitake Mushroom on Composition and Cariogenicity of Dental Plaque Microcosms in an In Vitro Caries Model

The aim of the current study was to investigate the anticariogenic potential of the (sub)fractions obtained from the edible mushroom shiitake (Lentinula edodes) in in vitro caries model. We used a modified constant depth film fermentor (CDFF) with pooled saliva as the inoculum and bovine dentin as a substratum. The test compounds were low molecular weight fraction (MLMW) of the shiitake extract and subfractions 4 and 5 (SF4 and SF5) of this fraction. Chlorhexidine (CHX) and water served as a positive and a negative control, respectively. Dentin mineral loss was quantified (TMR), microbial shifts within the microcosms were determined (qPCR), and the acidogenicity of the microcosms was assessed (CIA). From the compounds tested, the SF4 of shiitake showed strong inhibiting effect on dentin demineralization and induced microbial shifts that could be associated with oral health. The acid producing potential was increased, suggesting uncoupling of the glycolysis of the microbiota by the exposure to SF4. In conclusion, the results suggest that SF4 of shiitake has an anticariogenic potential.

Interspecies Interactions between Clostridium difficile and Candida albicans

Candida albicans and Clostridium difficile are two opportunistic pathogens that reside in the human gut. A few studies have focused on the prevalence of C. albicans in C. difficile-infected patients, but none have shown the interaction(s) that these two organisms may or may not have with each other. In this study, we used a wide range of different techniques to better understand this interaction at a macroscopic and microscopic level. We found that in the presence of C. albicans, C. difficile can survive under ambient aerobic conditions, which would otherwise be toxic. We also found that C. difficile affects the hypha formation of C. albicans, most likely through the excretion of p-cresol. This ultimately leads to an inability of C. albicans to form a biofilm. Our study provides new insights into interactions between C. albicans and C. difficile and bears relevance to both fungal and bacterial disease. ABSTRACT The facultative anaerobic polymorphic fungus Candida albicans and the strictly anaerobic Gram-positive bacterium Clostridium difficile are two opportunistic pathogens residing in the human gut. While a few studies have focused on the prevalence of C. albicans in C. difficile-infected patients, the nature of the interactions between these two microbes has not been studied thus far. In the current study, both chemical and physical interactions between C. albicans and C. difficile were investigated. In the presence of C. albicans, C. difficile was able to grow under aerobic, normally toxic, conditions. This phenomenon was neither linked to adherence of bacteria to hyphae nor to biofilm formation by C. albicans. Conditioned medium of C. difficile inhibited hyphal growth of C. albicans, which is an important virulence factor of the fungus. In addition, it induced hypha-to-yeast conversion. p-Cresol, a fermentation product of tyrosine produced by C. difficile, also induced morphological effects and was identified as an active component of the conditioned medium. This study shows that in the presence of C. albicans, C. difficile can persist and grow under aerobic conditions. Furthermore, p-cresol, produced by C. difficile, is involved in inhibiting hypha formation of C. albicans, directly affecting the biofilm formation and virulence of C. albicans. This study is the first detailed characterization of the interactions between these two gut pathogens. IMPORTANCE Candida albicans and Clostridium difficile are two opportunistic pathogens that reside in the human gut. A few studies have focused on the prevalence of C. albicans in C. difficile-infected patients, but none have shown the interaction(s) that these two organisms may or may not have with each other. In this study, we used a wide range of different techniques to better understand this interaction at a macroscopic and microscopic level. We found that in the presence of C. albicans, C. difficile can survive under ambient aerobic conditions, which would otherwise be toxic. We also found that C. difficile affects the hypha formation of C. albicans, most likely through the excretion of p-cresol. This ultimately leads to an inability of C. albicans to form a biofilm. Our study provides new insights into interactions between C. albicans and C. difficile and bears relevance to both fungal and bacterial disease.

Effects of Lactobacillus rhamnosus GG on saliva-derived microcosms

OBJECTIVE The probiotic strain Lactobacillus rhamnosus GG (LGG) is shown to hamper the presence of mutans streptococci in saliva and may have positive effects on oral health. We investigated the effects of LGG on the cariogenic potential and microbial composition of saliva-derived microcosms. DESIGN Single and dual species biofilms of LGG and Streptococcus mutans, and saliva-derived microcosms with or without LGG were grown in an Active Attachment Biofilm model. The microcosms were grown on bovine dentin/enamel discs in the presence or absence of sucrose (suc+/suc-). The presence of LGG was determined by multiplex ligation-dependent probe amplification (MLPA) and real-time PCR. Mutans streptococci (MS) and total viable counts, pH of the spent medium, capacity of lactate formation and integrated mineral loss in dentin was assessed. MLPA was used for identification and relative quantification of 20 oral microorganisms in the microcosms. Principal Component Analysis was applied to MLPA data. RESULTS LGG inhibited the growth of S. mutans in dual species biofilms and did not affect the pH. LGG established in saliva-derived microcosms and reduced MS counts significantly, but did not affect pH or dentin demineralization. Simultaneous growth of the microcosms with LGG under heavy cariogenic conditions (suc+) introduced a compositional shift in the microbial community. The CFU, real-time PCR and MLPA data correlated significantly. CONCLUSION We conclude that LGG established into and inhibited the growth of MS in complex saliva-derived biofilms, but this had no significant effect on cariogenic potential of the microcosms. This suggests that other microorganisms besides MS were responsible for increased cariogenicity of sucrose-exposed biofilms.

Novel metabolic activity indicator in Streptococcus mutans biofilms

Antimicrobial resistance of micro-organisms in biofilms requires novel strategies to evaluate the efficacy of caries preventive agents in actual biofilms. Hence we investigated fluorescence intensity (FI) in Streptococcus mutans biofilms constitutively expressing green fluorescent protein (GFP). Upon addition of glucose FI in these biofilms increased significantly to steady state levels. FI-increase could be inhibited by oral care products in a dose-responsive manner. Lactic acid produced in these biofilms was measured at the end of the FI-recording. A linear correlation with a coefficient of 0.96 (p<0.01) was observed between FI-increase and lactate production, irrespective of the inhibitor used. The viability of biofilm cells after chlorhexidine (CHX) titration was also examined. Reduction of FI-increase was observed at low concentrations of CHX whereas a loss in viability was only seen at high concentrations. In conclusion, GFP synthesis can be used as a metabolic activity indicator in S. mutans biofilms.

Effect of Vanadium chloroperoxidase on Enterococcus faecalis biofilms

INTRODUCTION The aim of this study was to explore the antimicrobial effect of vanadium chloroperoxidase (VCPO) reaction products on Enterococcus faecalis biofilms of 4 different strains. METHODS Twenty-four-hour biofilms of E. faecalis strains V583, ER5/1, E2, and OS-16 were incubated in mixtures with VCPO, halide (either bromide or chloride), and hydrogen peroxide. The antibacterial efficacy was assessed by colony-forming unit counts. RESULTS The VCPO reaction products had a similar efficacy in reducing the viability of the 4 strains of E. faecalis (94%; range, 87%-100%). Bromide as the halogen of choice was more effective on E. faecalis strains E2 and OS-16, as compared with chloride (Mann-Whitney U test; P < .05). Despite different quantities of produced biofilms by the 4 strains, VCPO treatment was similarly effective toward all strains (Kruskal-Wallis test; P < .05). CONCLUSIONS VCPO treatment results in an antimicrobial effect toward in vitro E. faecalis biofilms and might provide an addition to current endodontic treatment, possibly as an antimicrobial dressing.

Changes in the oral ecosystem induced by the use of 8% arginine toothpaste

OBJECTIVE Bacterial metabolism of arginine in the oral cavity has a pH-raising and thus, potential anti-caries effect. However, the influence of arginine on the oral microbial ecosystem remains largely unresolved. DESIGN In this pilot study, nine healthy individuals used toothpaste containing 8% arginine for eight weeks. Saliva was collected to determine arginolytic potential and sucrose metabolic activity at the Baseline, Week 4, Week 8 and after a two weeks Wash-out period. To follow the effects on microbial ecology, 16S rDNA sequencing on saliva and plaque samples at Baseline and Week 8 and metagenome sequencing on selected saliva samples of the same time-points was performed. RESULTS During the study period, the arginolytic potential of saliva increased, while the sucrose metabolism in saliva decreased. These effects were reversed during the Wash-out period. Although a few operational taxonomic units (OTUs) in plaque changed in abundance during the study period, there was no real shift in the plaque microbiome. In the saliva microbiome there was a significant compositional shift, specifically the genus Veillonella had increased significantly in abundance at Week 8. CONCLUSION Indeed, the presence of arginine in toothpaste affects the arginolytic capacity of saliva and reduces its sucrose metabolic activity. Additionally, it leads to a shift in the salivary microbiome composition towards a healthy ecology from a caries point of view. Therefore, arginine can be regarded as a genuine oral prebiotic.